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Transcription…. Transcription – the process of copying a DNA template into an RNA strand Accomplished via DNA dependent RNA polymerase (aka RNA polymerase) Similar to DNA replication but: uses RNA polymerase RNA product is displaced nearly immediately from template is less accurate

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transcription
Transcription…
  • Transcription – the process of copying a DNA template into an RNA strand
  • Accomplished via DNA dependent RNA polymerase (aka RNA polymerase)
    • Similar to DNA replication but:
    • uses RNA polymerase
    • RNA product is displaced nearly immediately from template
    • is less accurate
    • is not interested in the whole genome, only sections
transcription1
Transcription…
  • By the end of this series, you should be able to explain much of this animation
  • http://www.as.wvu.edu/~dray/219files/Transcription_588x392.swf
slide3
Bacterial RNA polymerase 50 - 100 nucleotides/sec
  • Most genes are transcribed simultaneously by numerous polymerases
  • Polymerase moves along DNA in 3' —> 5' direction
  • Complementary RNA constructed in ____ direction
    • RNAn + NPPP —> RNAn+1 + PPi
transcription2
Transcription
  • How does the polymerase know where to start?
    • Promoter = the assembly point for the transcription complex
  • RNA polymerases cannot recognize promoters on their own - transcription factors
    • Transcription factors - enzymes have evolved to recognize (physically interact with) specific DNA sequences and with other proteins
slide5
Prokaryotic Gene Regulation
  • Preliminaries
  • DNA binding proteins
  • Minor vs. Major grooves
  • Bases are exposed in the grooves, providing binding sites
  • Each bp provides characteristic binding sites

A – H acceptor, D – H donor, H – nonpolar H, M – methyl group

slide6
Prokaryotic Gene Regulation
  • Protein-nucleic acid binding
  • Most proteins to be discussed bind specific DNA sequences
  • Most commonly via α-helix insertion into major groove(s)
    • H donor/acceptors
    • + charge on helix interaction with phosphate backbone
  • Helix-turn-helix motif
      • 1st DNA binding domain identified
slide7
Transcription
  • Three-phase progression
  • Initiation –
    • Promoter recognition and binding
    • DNA melting
    • Transcript initiation
  • Elongation
    • mRNA produced
  • Termination
    • Polymerase and RNA released
slide8
Transcription
  • Bacterial transcription
  • Promoters
  • Core enzyme (α2ββ’ω) will begin transcription just about anywhere
  • σ provides specificity
  • Core + σ = holoenzyme
  • E. coli  σ70
  • σ70 promoters
    • 2 conserved sequences of 6 bp each
    • -10, -35
slide9
Transcription
  • Bacterial transcription
  • Promoters
  • Not all -10 and -35 sequences are identical
  • Consensus sequences
    • -35 – TTGACA
    • -10 - TATAAT
transcription3
Transcription
  • Prokaryotic Transcription
  • Bacterial promoters are located just upstream of the RNA synthesis initiation site
    • The nucleotide at which transcription is initiated is called +1; the preceding nucleotide is –1
    • DNA preceding initiation site (toward template 3' end) are said to be upstream
    • DNA succeeding initiation site (toward template 5' end) are said to be downstream
transcription4
Transcription
  • Prokaryotic Transcription
  • One RNA polymerase with 5 subunits tightly associated to form core enzyme
  • Core enzyme minus sigma (σ) factor will bind to any DNA.
    • By adding σ, RNA pol will bind specifically to promoters (-10 & -35 sequences)
  • Transcription begins de novo
  • Requires stable interaction b/t base and template while second base is recruited
slide12
Transcription
  • Bacterial transcription
  • Three models of initial transcription
  • How to explain?
  • Transient excursion – Pol moves along DNA chain
  • Inchworming –
  • Scrunching – DNA pulled in
  • Experiments demonstrate:
    • Pol remains stationary on promoter
    • Pol subunits remain stationary relative to one another
  • Suggests that ‘scrunching’ is it
    • As DNA/RNA ‘piles up’ inside the polymerase, it creates pressure which contributes to forcing the polymerase off of the promoter
slide13
Transcription
  • Bacterial transcription
  • Several false starts before elongation begins
    • ≤ 9bp
  • ‘Escape’ - transcription of 10+ bp leading to elongation phase
  • 10+ bp too long to remain hybridized to tempate
  • Pol must break interactions with promoter and regulatory factors
  • σ ¾ linker must be ejected from RNA exit channel
  • ‘Scrunched’ DNA likely provides the energy to break pol-promoter interactions and dislodge σ
slide14
Transcription
  • Bacterial transcription
  • Termination
  • Rho (ρ) dependent
  • ATPase activity leads to translocation along transcript
slide15
Transcription
  • Bacterial transcription
  • Termination
  • ρ-dependent
  • Rut (ρ utilization) sites – not well-defined but sequence dependent
  • http://archive.microbelibrary.org/microbelibrary/files/ccImages/Articleimages/sandrin/rhodependent.htm
  • http://highered.mcgraw-hill.com/sites/dl/free/0072835125/126997/animation21.html
slide16
Transcription
  • Bacterial transcription
  • Termination
  • ρ-independent
  • Two sequence elements
    • Inverted repeat
    • ~8 A:T stretch
  • Hairpin formation disrupts polymerase function
  • A:U binding weak
transcription5
Transcription
  • Eukaryotic vs. Prokaryotic Transcription
  • Much of what we know is derived from studies of RNA pol II from yeast
    • 1. Seven more subunits than its bacterial RNA pol
    • 2. The core structure & the basic mechanism of transcription are virtually identical
    • 3. Additional subunits of eukaryotic polymerases are thought to play roles in the interaction with other proteins
    • 4. Eukaryotes require a large variety of accessory proteins or transcription factors (TFs)
slide18
Transcription
  • Eukaryotic transcription
  • Contrasts with prokaryotes
    • Three polymerases
      • RNA polymerase I (RNA pol I) - synthesizes the larger rRNAs (28S, 18S, 5.8S)
      • RNA polymerase II (RNA pol II)- synthesizes mRNAs & most small nuclear RNAs (snRNAs & snoRNAs)
      • RNA polymerase III (RNA pol III) - synthesizes various small RNAs (tRNAs, 5S rRNA & U6 snRNA)
    • Multiple general transcription factors (GTFs)
    • Additional regulatory elements (enhancers, chromatin modifiers, silencers, insulators, etc.)
  • RNA pol II – transcribes mRNA
slide19
Transcription
  • Eukaryotic transcription
  • RNA pol II core promoters
  • Four different sequence elements
    • TFIIB recognition element (BRE box)
    • TATA box
    • Initiator box (Inr)
    • Downstream promoter elements (DCE, DPE, MTE)
  • Various combinations in most Pol II promoters
    • All not necessary
    • Inr most common
slide20
Transcription
  • Eukaryotic transcription
  • Preinitiation complex formation
  • TBP component of TFIID
  • β sheet inserted into minor groove, bending DNA 80°
  • Specificity via phenylalanine chain intercalation on flanks of sequence
slide21
Transcription
  • Eukaryotic transcription
  • Preinitiation complex formation
  • TFIID TFIIA TFIIB TFIIF TFIIE TFIIH
slide22
Transcription
  • Eukaryotic transcription
  • Preinitiation complex formation
  • TFIID – initial binding and recruitment
  • TFIIA – clamp
  • TFIIB - recruitment of pol II, may insert into RNA exit channel
  • TFIIF – stabilize complex, required for recruitment of TFIIE/H
  • TFIIE – recruit and regulate TFIIH
  • TFIIH – kinase
    • Largest GTF
    • Phosphorylates carboxy-terminal domain (CTD) of pol II
    • http://www.crocoduck.bch.msstate.edu/BCH4713/Transcription.wmv
rna processing mrna
RNA processing – mRNA
  • 5’ cap
    • The raw transcript will be immediately degraded in the cytoplasm so it must be marked and protected
  • Possible/known functions of 5’ cap
    • May prevent exonuclease digestion of mRNA 5' end,
    • Aids in transport of mRNA out of nucleus
    • Important role in initiation of mRNA translation
rna processing mrna1
RNA processing – mRNA
  • mRNA processing – Splicing
  • Requires break at 5' & 3' intron ends (splice sites) & covalent joining of adjacent exons (ligation)
      • http://www.as.wvu.edu/~dray/219files/mRNASplicingAdvanced.wmv
  • Why introns?
    • Disadvantages – extra DNA, extra energy needed for processing, extra energy needed for replication
    • Advantages – modular design allows for greater variation and relatively easy introduction of that variation
rna processing mrna2
RNA processing – mRNA
  • mRNA processing – Splicing
  • Splicing MUST be absolutely precise
  • Most common conserved sequence at eukaryotic exon-intron borders in mammalian pre-mRNA is G/GU at 5' intron end (5' splice site) & AG/G at 3' end (3' splice site)
rna processing mrna3
RNA processing – mRNA
  • mRNA processing – Splicing
  • Sequences adjacent to introns contain preferred nucleotides that play an important role in splice site recognition
rna processing mrna4
RNA processing – mRNA
  • mRNA processing – Splicing
  • Nuclear pre-mRNA (common)
    • snRNAs + associated proteins = snRNPs
      • snRNAs – 100-300 bp
      • U1, U2, U4, U5, U6
    • 3 functions for snRNPs
      • Recognize sites (splice site and branch point site)
      • Bring these sites together
      • Catalyze cleavage reactions
    • Splicosome – the set of 5 snRNPs and other associated proteins
    • Summary movie available at:
    • http://www.as.wvu.edu/~dray/219files/mRNAsplicing.swf
rna processing mrna5
RNA processing – mRNA
  • mRNA processing – Splicing
  • 1. U1 and U2 snRNPs bind via complementary RNA sequences
  • Note the A bulge produced by U2
  • U2 is recruited by proteins associated with an exon splice enhancer (ESE) within the exon
rna processing mrna6
RNA processing – mRNA
  • mRNA processing – Splicing
  • 2. U2 recruits U4/U5/U6 trimer
  • U6 replaces U1, U1 and U4 released
  • U5 binds to upstream exon
rna processing mrna7
RNA processing – mRNA
  • mRNA processing – Splicing
  • 3. U6 catalyzes two important reactions
    • Cleavage of upstream exon from intron (bound to U5)
    • Lariat formation with A bulge on intron
  • Exons are ligated
  • U2/U5/U6 remain with intron
slide31
DNA/RNA Structure

OH

OH

NB

NB

NB

NB

DNA

RNA

OH

O

OH

OH

P

O

C

OH

O

P

P

P

O

O

O

C

C

C

O

OH

OH

rna processing mrna8
RNA processing – mRNA
  • mRNA processing – Splicing
  • http://www.crocoduck.bch.msstate.edu/BCH4713/ch13_group_II_introns.html
  • Several lines of evidence suggest that it is the RNA in the snRNP that actually catalyzes the splicing reactions
    • 1. Pre-mRNAs are spliced by the same pair of chemical reactions that occur as group II (self-splicing) introns
    • 2. The snRNAs needed for splicing pre-mRNAs closely resemble parts of the group II introns
  • Proteins likely serve supplemental functions
    • 1. Maintaining the proper 3D structure of the snRNA
    • 2. Driving changes in snRNA conformation
    • 3. Transporting spliced mRNAs to the nuclear envelope
    • 4. Selecting the splice sites to be used during the processing of a particular pre-mRNA
slide33
Transcription
  • Polyadenlyation
  • The poly(A) tail – 3' end of most mRNAs contain a string of adenosine residues (100-250) that forms a tail
    • Protects the mRNA from degradation
    • AAUAAA signal ~20 nt upstream from poly(A) addition site
    • Poly(A) polymerase, poly(A) binding proteins, and cleavage factors are involved
    • Simple animation on website
slide34
Transcription
  • Eukaryotic transcription
  • Termination
  • Torpedo model
  • Post-cleavage RNA is uncapped
  • Recognized by Rnase (Xrn2 in humans)
  • Again, CTD bound
  • Highly processive, displaces Pol II
slide35
Transcription
  • Eukaryotic RNA pol I
  • rRNA transcription
  • SL1 = TBP + three TAFs
slide36
Transcription
  • Eukaryotic RNA pol III
  • tRNA and 5S transcription
  • Internal promoter sequences
slide37
Prokaryotic Gene Regulation
  • Principles
  • Activators and repressors
  • DNA binding proteins
  • Primary level of action – transcription initiation
  • Activators enhance RNA polymerase binding
  • Repressors block RNA polymerase binding
  • Many regulatory proteins work via allostery
slide38
Prokaryotic Gene Regulation
  • Principles
  • Action at a distance
  • Interaction b/t distantly binding proteins accommodated by DNA loops
  • Often aided by architectural proteins
slide39
Prokaryotic Gene Regulation
  • Control of Gene Expression: Prokaryotes
    • The operon - in bacteria, genes for enzymes of metabolic pathway are usually clustered in functional complex under coordinate control
    • Terminology:
    • 1. Genes - code for operon enzymes; usually adjacent to each other; turn on one, turn on all
    • 2. Promoter
    • 3. Operator – typically resides adjacent to or overlapping with the promoter; repressor protein binding site
    • 4. Repressor - gene regulatory protein; binds with high affinity to operator
    • 5. Regulatory gene - encodes repressor protein
slide40
Prokaryotic Gene Regulation
  • The operon…
    • The repressor is key to operon expression; if it binds to operator; it shields promoter from polymerase & prevents transcription
      • 1. Repressor binding to operator depends on conformation, which is regulated by a key compound in the metabolic pathway (lactose or tryptophan)
      • 2. Concentration of key metabolite determines if operon is active or inactive at any given time
slide41
Prokaryotic Gene Regulation
  • The lac operon…
    • An inducible operon – the presence of a key substance induces the transcription of the genes.
    • Regulates production of the enzymes needed to degrade lactose in bacterial cells
      • Genes in the lac operon
      • 1. z gene - encodes β-galactosidase
      • 2. y gene - encodes galactoside permease; promotes lactose entry into cell
      • 3. a gene - encodes thiogalactoside acetyltransferase; its physiological role is unclear
slide42
Prokaryotic Gene Regulation
  • Prokaryotic gene expression…
    • Lactose (disaccharide) - made of glucose & galactose
    • Oxidation provides the cell with metabolic intermediates & energy
    • The β-galactoside linkage is broken in the first step of catabolism - β-galactosidase
slide43
Prokaryotic Gene Regulation
  • Control of Gene Expression: Prokaryotes
    • Prokaryotes live in constantly changing environment
    • It is advantageous for cells to use available resources in most efficient way so regulate responses
    • Thus, they respond by selective gene expression
    • If lactose is absent—> β-galactosidase not needed & not present (<5 copies of enzyme, 1 of the corresponding mRNA)
    • If lactose is present —> enzyme levels rise ~1000-fold in a few minutes; lactose has induced the synthesis of β-galactosidase
slide44
Prokaryotic Gene Regulation
  • The lac operon…
    • 1. If lactose is present in medium, it enters cell, binds lac repressor, changing its shape. Lactose acts as an inducer
    • 2. Lactose-bound repressor cannot bind operator DNA
    • 3. If lactose levels fall, it dissociates from repressor, changing repressor back to active shape
    • 4. Repressor binds operator and physically blocks polymerase from reaching structural genes, turns off transcription
    • Lac operon movie
slide45
Prokaryotic Gene Regulation
  • The lacoperon
  • Lac repressor binding
slide46
Prokaryotic Gene Regulation
  • The lacoperon
  • Both bind using similar amino acid motifs
  • Helix-turn-helix
  • Recognition helix interacts with DNA
  • λ-repressor example
slide47
Transcription...
  • Control of Gene Expression: Prokaryotes
    • Tryptophan - essential amino acid needed for protein synthesis; if it is not in the growth medium, it must be produced by bacterium
      • 1. In its absence, cells contain enzymes & their mRNAs needed to make tryptophan
      • 2. If tryptophan is available in medium, bacteria don't need enzymes to make it; the genes of those enzymes are repressed within a few minutes & the production of the enzymes stops
slide48
Transcription...
  • The trp operon…
    • A repressible operon – the presence of a key substance represses the transcription of genes.
    • Repressor is active only if bound to specific factor which functions as a co-repressor (like tryptophan)
slide49
Transcription...
  • The trp operon…
    • 1. Without tryptophan, operator site is open to binding by RNA polymerase
    • 2. Production of enzymes that synthesize tryptophan
    • 3. When tryptophan is available, enzymes of tryptophan synthetic pathway are no longer needed
    • 4. Increased tryptophan concentration leads to formation of tryptophan-repressor (active repressor)
    • 5. Repressor binds DNA at operator, blocks transcription
    • http://bcs.whfreeman.com/thelifewire/content/chp13/1302002.html
slide50
The Cell Nucleus…
  • Control of Gene Expression: Eukaryotes
    • A single human cell contains enough DNA (6 billion bp) to encode several million different polypeptides
      • 1. Most of this DNA does not actually code for proteins, mammalian genomes are thought to contain ~30,000 protein-coding genes
      • 2. A typical mammalian cell may make ~5,000 different polypeptides at any given time
      • 3. Many of these are made by virtually all cells of the organism
      • 4. Cells also make proteins unique to its differentiated state; giving the cell its unique characteristics
      • 5. Regulating eukaryotic gene expression is an extremely complex process, just starting to be understood
slide51
The Cell Nucleus…
  • Control of Gene Expression: Eukaryotes
    • Three levels of control
      • Trascriptional
      • Processing
      • Translational
slide52
The Cell Nucleus…
  • Regulatory Regions
    • The regulatory region of a gene can be thought of as an integration center for that gene's expression
      • The extent to which a given gene is transcribed depends upon particular combination of TFs bound to its upstream regulatory elements
        • 1. Roughly 5 – 10% of genes encode TFs
        • 2. Thus, a nearly unlimited number of possible combinations of interactions among TFs is possible
        • 3. Complexity of interactions is revealed in marked variation in gene expression patterns between cells of different type, different tissue, different developmental stage & different physiological state
slide53
The Cell Nucleus…
  • Regulatory Regions
    • Promoter elements – regions upstream of a gene that regulate the initiation of transcription.
    • Most eukaryotic promoter elements can be roughly divided in to ‘proximal’ and ‘distal’
    • Proximal promoter elements (-50 to -200bp):
      • TATA box –
        • Consensus sequence – TATAAA
        • Usually at ~-30
      • CAAT box –
        • Consensus sequence – CAAT
        • Usually ~-70
      • GC box –
        • Consensus sequence – GGGCGG
        • Often multiple copies within 100 bp upstream of start codon
slide54
The Cell Nucleus…
  • Regulatory Regions
    • Proximal promoter elements (-50 to -200bp):
      • TATA box –
        • Site of assembly of the transcription complex:
        • RNA polymerase II, all necessary transcription factors
      • CAAT box and GC box –
        • Regulate the frequency of transcription via binding of transcription factors
slide55
The Cell Nucleus…
  • More Regulatory Elements
    • Enhancers
    • Raise transcription rates above the basal level
      • 1. Have a unique property: they can be moved experimentally from one place to another within a DNA molecule (even be inverted) without affecting the ability to stimulate transcription
      • 2. Deletion of an enhancer can decrease the level of transcription by 100-fold or more
      • 3. Some enhancers are located thousands or even tens of thousands of base pairs upstream or downstream from the gene whose transcription they stimulate
slide56
The Cell Nucleus…
  • More Regulatory Elements
    • Enhancers
    • Thought to stimulate transcription by influencing events that occur at core promoter
      • A. Enhancers & core promoters can be brought together via DNA loops
      • Remember movie from chapter 11?
      • B. What prevents enhancer from binding to inappropriate promoter located even farther downstream?
        • 1. insulators
        • 2. insulator sequences may bind to proteins of nuclear matrix; DNA segments between insulators correspond to looped domains of chromatin
    • How do transcriptional activators bound at enhancer stimulate transcription initiation at core promoter?
      • coactivators; 2 basic types:
        • 1. Those that interact with components of the basal transcription machinery (general TFs & RNA polymerase II) - lead to assembly of preinitiation complex & initiation of RNA synthesis
        • 2. Those that act on chromatin, converting it from a state relatively inaccessible to transcription machinery to a much more transcription-friendly state
slide57
The Cell Nucleus…
  • Control of Gene Expression: Eukaryotes
    • Transcriptional control is orchestrated by actions of a large number of proteins called transcription factors (TFs);
    • 1. General TFs - bind at core promoter sites in association with RNA polymerase
    • 2. Sequence-specific TFs - bind to various regulatory sites of particular genes; they either stimulate (transcriptional activators) or inhibit (transcriptional repressors) transcription of adjacent genes
slide58
Eukaryotic Gene Regulation
  • DNA binding domains
  • Dimerization common
  • Bacterial and eukaryotic DNA binding domains can often be interchanged
  • Homeodomains
    • Helix-turn-helix
    • All eukaryotes
slide59
Eukaryotic Gene Regulation
  • Zinc binding domains
  • Zinc finger/zinc cluster
    • Cys/His
    • TFIIIA
    • Glucocorticoid receptor

TFIIIA

slide60
Eukaryotic Gene Regulation
  • Helix-loop-helix
  • Long helix for DNA binding
  • Shorter helix for dimerization or other function
slide61
Eukaryotic Gene Regulation
  • HMG box
  • Three helices in boomerang shape
  • DNA bending
  • Architectural factors
  • SRY gene and testosterone and XY females
  • HMG factor binds upstream of SRY to increase testosterone production
  • Activation domains
  • Not well-defined
  • Often grouped by amino acid content
slide62
Eukaryotic Gene Regulation
  • Signal transduction
  • any process by which a cell converts one kind of signal or stimulus into another
  • Often in response to external stimuli
  • Many, many different pathways specific to particular situations
  • Some general commonalities:
    • Ligand usually initiates the pathway
    • Cell surface receptors
    • Path is often a cascade of kinases
    • Sometimes, activating region is “unmasked”
    • Sequestering inactive activators and repressors in the cytoplasm
    • Sometimes, participants come in pieces that are later combined
  • Examples follow
slide63
Eukaryotic Gene Regulation
  • Signal transduction
  • Example – STAT (signal transducer and activator of transcription)
  • Cytokine – intercellular signaling molecule
  • SH2 domain of STAT is variable and different STATs will target different genes
  • Three examples on website
slide64
Eukaryotic Gene Regulation
  • Signal transduction
  • Example – Ras/MAPK (mitogen activated protein kinase)
  • GDP-GTP exchange induces conformational change in Ras
  • Kinase cascade with MAPK proteins
  • Example animations on website (with cool sound effects)
slide65
Eukaryotic Gene Regulation
  • Signal transduction
  • Example – A generalized cAMP pathway
    • G protein
      • Inactive – trimer bound to GDP
      • Activation - GDP replaced by GTP
      • Active – monomer + GTP, dimer
      • Largest superfamily of human proteins (1000+)
    • cAMP – a common second messenger generated by adenylatecyclase
    • Serine-Threoninekinase - phosphorylate serine or threonine in the affected polypeptide

cAMP

Regulatory

subunits

Catalytic

subunits

cAMP response

element binding

(TGANNTCA)

slide66
Eukaryotic Gene Regulation

Tumor Necrosis

Factor

  • Signal transduction
  • Example – Sequestration of inactive transcription factor

TNF Receptor

Associated Factor 2

IKB kinase

Inhibitor

of KB

Nuclear Factor

kappa-light-chain-

enhancer of

activated B cells

slide67
Eukaryotic Gene Regulation
  • Signal transduction
slide68
The Cell Nucleus…
  • Post-transcriptional control
    • Alternative splicing – a single gene can encode two or more related proteins; multiple processing pathways for the transcript
      • Genes of complex plants & animals have numerous introns & exons —> use a different exon combination, get a different protein
      • Roughly 40 – 60% of human genes are subject to alternate splicing
slide69
The Cell Nucleus…
  • Post-transcriptional control
  • Translational-level control
  • 3 aspects of translational-level control
    • A. Localization of mRNAs to certain sites within a cell
    • B. Controlling whether or not an mRNA is translated and, if so, how often
    • C. Controlling the half-life of the mRNA, a property that determines how long the message is translated
  • Mechanisms usually work via interactions between mRNAs & cytoplasmic proteins
slide70
The Cell Nucleus…
  • Post-transcriptional control
    • mRNAs contain noncoding segments, called untranslated regions (UTRs) at both their 5' & 3' ends; these are sites where most translational control is effected
      • 1. 5' UTR extends from methylguanosine cap at start of message to AUG initiation codon
      • 2. 3' UTR extends from termination codon at end of coding region to the end of the poly(A) tail attached to nearly all eukaryotic mRNAs
slide71
The Cell Nucleus…
  • Translational-level control…
  • Cytoplasmic localization of mRNAs
    • Example: the fruit fly, anterior-posterior axis
      • 1. Axis formation is influenced by localization of specific mRNAs along same axis in the oocyte
      • 2. Bicoid mRNAs preferentially localized at anterior end; oskar mRNAs preferentially localized at opposite end
      • 3. Protein encoded by bicoid mRNA is critical for head & thorax development; oskar protein is required for formation of germ cells, which develop at posterior end of larva
      • 4. Localizing mRNAs is more efficient than localizing their corresponding proteins, since each mRNA can be translated into large numbers of protein molecules
slide72
The Cell Nucleus…
  • Translational-level control…
  • Cytoplasmic localization of mRNAs
    • 3' UTR governs localization of bicoid & oskar mRNAs
      • 1. Join foreign gene coding region to DNA sequence encoding 3’ UTR of oskar or bicoid
      • 2. Place in fruit flies & see what happens when the foreign gene is transcribed during oogenesis —> foreign gene goes to site determined by its 3’ UTR
      • 3. Localization of mRNAs is mediated by specific proteins that recognize mRNA localization sequences (zipcodes) in this region of mRNA
slide73
The Cell Nucleus…
  • Translational-level control…
  • Controlling mRNA translation
  • Example: mRNAs stored in unfertilized egg are templates for proteins synthesized during the early stages of development;
    • rendered inactive by association with inhibitory proteins
    • Activation of these stored mRNAs involves at least two distinct events:
      • 1. Release of bound inhibitory proteins
      • 2. Increase in length of poly(A) tails by action of an enzyme residing in egg cytoplasm
slide74
The Cell Nucleus…
  • Translational-level control…
  • Controlling mRNA stability
  • The longer an mRNA is present in cell, the more times it can serve as template for polypeptide synthesis
    • c-fos mRNA made in response to changes in external conditions in many cells; degraded rapidly in cell (half-life of 10 - 30 min); involved in cell division control
    • In contrast, dominant cell protein mRNAs in a particular cell, like those for hemoglobin, (half-life >24 hours)
slide75
The Cell Nucleus…
  • Translational-level control…
  • Controlling mRNA stability
  • mRNA longevity is related to length of poly(A) tail
    • 1. Early study - mRNAs lacking poly(A) tails are rapidly degraded after injection into cell, whereas same mRNA with poly(A) tail is relatively stable
    • 2. Typical mRNA has ~200 adenosine residues when it leaves nucleus
    • 3. Gradually reduced in length as it is nibbled away by poly(A) ribonuclease
    • 4. No effect until the tail is reduced to ~30 A residues; once shortened to this length, the mRNA is usually degraded rapidly
slide76
The Cell Nucleus…
  • Translational-level control…
  • Controlling mRNA stability
  • Tail length not the whole story;
    • mRNAs starting with same size tail have very different half-lives –
    • 3' UTR plays role
    • 3'-UTR of α-globin mRNA contains a number of CCUCC repeats that serve as binding sites for specific proteins that stabilize mRNA; if these sequences are mutated, the mRNA is destabilized
    • Short-lived mRNAs often contain destabilizing sequences (AU-rich elements; AUUUA repeats) in their 3' UTR; thought to bind proteins that destabilize mRNA
slide77
The Cell Nucleus…
  • Post-translational control…
  • Controlling protein stability
  • Every protein is thought to have characteristic longevity (half-life) or the period of time during which it has a 50% likelihood of being destroyed
    • A. Some enzymes (those of glycolysis or erythrocyte globin molecules) are present for days to weeks
    • B. Other proteins required for a specific, fleeting activity (regulatory proteins that initiate DNA replication or trigger cell division) may survive only a few minutes
    • C. All of the proteins, regardless of expected survival time, are degraded by proteasomes
    • D. Factors controlling a protein's lifetime are not well understood
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